Vertebrate genes are typically split into a number of small exons separated by considerably larger introns. This proposal is aimed at investigating the mechanism whereby exons are recognized and assembled into the active spliceosome so as to correctly orchestrate constitutive and differential splicing. The hypothesis upon which this proposal rests is that the exon is the unit of splice site recognition in vertebrates (in contrast to the intron in S. cerevisiae) in a process that we have referred to as exon definition. During the next period we plan to continue to investigate the merits of an exon perspective of splice site recognition. Five questions will be posed: 1. What cis-acting sequences determine the efficiency of exon definition? We will ask what combination of splice site strength, exon length, and exon sequence are optimal for exon definition. The hypothesis to be tested is that exon strength is a combination of these elements, and that constitutive exons maintain a minimal balance of all three for recognition; and that alternative exons will be sub-optimal for one or more to permit regulation. We are especially interested in investigating the possibility that certain sequences internal to exons promote or inhibit their recognition. 2. How are constitutive long exons recognized? Exons in vertebrate genes rarely exceed 300 nucleotides and our published work suggests that exon definition has operational limits in this range. Occasional long exons, however, exist. We will ask how they are recognized. 3. How are constitutive mini-exons recognized? Some vertebrate exons are exceedingly small (under 12 nucleotides). Exon definition suggests that both ends off an exon are simultaneously recognized, a problematic situation for such short exons. We will ask how mini-exons are recognized. 4. What is the molecular mechanism of exon definition? To gain an understanding of how exon definition operates we will determine the composition of the exon definition complex. We will also investigate the role of ATP in exon definition? 5. Can we establish a complementation system for exon definition factors? To study the factors required for exon definition, we wish to establish an extract that is negative for exon definition to which we can add fractions that restore exon definition. This complementation system will be used to decipher the role of individual factors.